The acute shortage of human organs for transplantation has stimulated exploration into the possibility of using non-human donor organs for xenotransplantation. Pigs are now regarded as the most likely species to serve as donors for clinical xenotransplantation. The first major immunological barrier to overcome to allow successful xenotransplantation is rejection by antibodies present in humans that are directed toward the carbohydrate epitope Galalpha1-3Galbeta1-4G1cNAc-R, (alphaGal) on porcine tissues. A mouse model has been developed in this laboratory to test whether the establishment of molecular chimerism through genetic engineering of bone marrow can prevent production of alphaGal reactive antibodies. Knockout mice (GT0 mice) lacking the gene encoding alpha(1-3)galactosyltransferase (alphaGT) develop serum antibodies reactive against alphaGal. Bone marrow cells harvested from GT0 mice were infected with retroviruses carrying the gene encoding porcine alphaGT, or control viruses carrying only the neomycin (NEO) resistance gene. Lethally-irradiated (10.25Gy) GT0 mice were then reconstituted with either alphaGT or NEO transduced BMC. While control mice reconstituted with NEO transduced BM developed alphaGal reactive serum antibodies, mice receiving alphaGT transduced BMC did not develop alphaGal reactive antibodies over a 25 week follow-up period. These results suggest that expression of a retrovirally transduced alphaGT gene in bone marrow derived cells may prevent the generation of B cells producing alphaGal reactive antibodies. The specific aims are to: 1) Determine the long-term effect of genetic engineering of bone marrow on production of alphaGal reactive antibodies, and whether the establishment of molecular chimerism is sufficient to prevent humoral rejection of transplanted organs; and 2) Determine the mechanism by which expression of the retrovirally transduced alphaGT gene in bone marrow derived cells prevents development of alphaGal reactive antibodies. These studies should provide practical information important for the field of xenotransplantation, the application of gene therapy to induce B cell tolerance, and may also advance our understanding of self-nonself discrimination in shaping the B cell repertoire. Furthermore, these studies may generally applicable for re-establishing B cell tolerance in auto-immune individuals.